A unique disulfide bond between C110 and C187 is critical for rhodopsin structure. Mutants of intradiscal loops, and some of the autosomal dominant retinitis pigmentosa (ADRP) mutants are believed to be defective in folding resulting in the lack of S-S bond formation. Our long term goal is to understand structure-function relationship in bovine rhodopsin by expression in COS cells and characterization of the mutant proteins. This proposal is aimed at: 1. investigating the molecular basis of defects caused by the absence of the S-S bond. 2. identifying Light induced changes in transmembrane helices C and F of opsin. We will examine directly in the membranes, the ability of mutants C110S & C187S to bind the chromophore 11 -cis-retinal, by UV/Visible difference spectroscopy and by crosslinking of the 3H-retinal to opsin. We will examine whether or not a noncovalent interaction between residues 110-187 suppresses the defect. 1-3 nmoles of mutant proteins can be obtained from transfected COS cells. Retinal binding to wildtype and unglycosylated opsin can be measured by spectroscopy and crosslinking techniques. In a mutant, CllOE-Cl87R, a blue shifted chromophore with low yield was observed possibly due to an ion pair interaction of the introduced residues. A systematic study of ion pair replacement of the S-S bond will help us establish the mechanism by which stabilization of the retinal pocket takes place. To identify the defect in some of the ADRP mutations, substitution of amino acids with different side chain characters will be made at seven ADRP sites. The location, post-translational modification and the function of the mutant proteins expressed in COS cells will be examined. The number of S-S bonds in the expressed defective proteins will be measured. We have optimized a sensitive technique to measure the number of S-S bonds in opsin, based on the specific cleavage of disulfide bond by stoichiometric incorporation of 14CN. The wild type opsin from the COS cells and the ROS, incorporate lmol/mol CN. The defective mutants are expected to incorporate substoichiometric or none, if a defect in S-S bond formation indeed exists in them. The out come of this test will help us to understand the molecular basis of a heterogeneous degenerative neuronal disorder. Molecular modelling studies show that transmembrane helices C and F of rhodopsin interact closely with retinal, and therefore might be primarily responsible for transduction of signal across the membrane. To examine this we will create mutants with-two amino acid insertions in helices C & F and examine their ability to activate transducin in response to light. Contact of retinal with specific residues is expected to be disrupted by insertion and result in the loss of transducing ability. In another approach we will introduce cysteines at the cytoplasmic border of helices C & F. The solvent accessibility of the introduced cysteines in dark and light activated mutant proteins will be studied. This will be important to explain transduction in terms of intramolecular changes.